Multicolumn sequential separation process

ABSTRACT

A process is disclosed for separating biomolecules from an aqueous solution containing the biomolectules and impurities, having different affinities and/or interactions with a solid support. The solution is passed over a fixed bed of chromatographic resin containing at least three zones, with flow of liquid being arranged between adjacent zones and between a last and first zone. Each of several sequences includes at least an adsorption stage, a rinsing stage, or a desorption stage, with each subsequent sequence being carried out by a downstream displacement of fronts in the zones by approximately the same increment before the periodical displacement of the introduction and withdrawal points.

REFERENCE TO RELATED APPLICATIONS

This application is a 371 of PCT/FR07/01012 filed Jun. 18, 2007.

FIELD OF THE INVENTION

A subject of the present invention is a multicolumn sequentialadsorption separation process. The invention is applicable generally toany separation involving an interaction between a product of interestand a stationary phase. The invention applies in particular to thepurification of molecules of interest, in particular biomolecules,proteins, antibiotics, amino acids, polynucleotides and polypeptides andother biological and chemical molecules.

PRIOR ART

The chromatography (in general liquid chromatography) purificationprocesses are characterized by bringing one or more liquid phase(s)(called mobile phase(s)) into contact with a solid phase (called astationary phase). The product of interest injected in the liquid phaseproduces one or more interactions of various kinds with the stationaryphase depending on the chromatographic separation technique selected:normal or reversed-phase chromatography, ion exchange, affinity,size-exclusion, hydrophobic interaction, etc. chromatography. Itsdisplacement within the chromatographic device is therefore differentfrom the displacement of the other products contained in the feed to betreated. Based on this difference in interaction, it is possible topurify or enrich one of the fractions in the molecule of interest.

For example, the production of an amino acid, such as lysine, byfermentation leads to an aqueous culture broth containing the dissolvedamino acid (for example lysine), microorganisms and impuritiesessentially consisting of residues from substrates and from cellularmetabolism of said microorganisms. Similarly, the lysine may also beavailable in combination with other elements in an aqueous solution,which has to be purified in order to extract the lysine. The amino acidsand in particular lysine are food supplements commonly used in theagri-food industry for livestock. Thus, the separation of solutionscontaining lysine, or any other amino acid, can be carried out by achromatographic technique, in this case on an ion exchange resin.

In the area of the purification of biomolecules, the target molecule iscontained in a complex mixture, obtained for example from a fermentationmedium (as in the case of lysine), or from a solution extracted fromanimal or plant tissues. The target molecule is present at lowconcentration and in many cases it is not the main species. Purificationof these complex media uses the chromatographic separation techniquesmentioned above.

The stages that make up a process of purification can be implemented invarious ways, the most conventional being the so-called batch mode inwhich each of the stages takes place successively in a single columnconsisting of the stationary phase. In order to improve thepossibilities of separation between the numerous constituents that arepresent, in this batch mode it is possible to use different eluents insuccession. The latter make it possible to vary the distributioncoefficients of the various compounds between the stationary phase andthe eluting liquid phase. Thus, a species that would have a strongaffinity for the stationary phase under certain elution conditions canlose this affinity entirely if the elution conditions are changed. Thischromatographic technique using several eluents requires a succession ofstages. It makes it possible to apply a principle of separation byadsorption and then desorption. It permits the extraction of the desiredmolecule from a mixture or the purification of a solution by removing anunwanted impurity.

For example, the ion exchange purification processes on a solid support,activated carbon or resin, are characterized by a succession of stages,repeated cyclically, which can include a stage of fixation, of elution,and of regeneration. In certain cases there may be several regenerationand rinsing phases. Based on said batch process, the most robustsolution for ensuring continuous production is to have at least twocolumns, one in production, while the other successively undergoes allthe stages of regeneration. Production is then continuous but thevarious flows produced during regeneration are not. It is the quest forcontinuous availability and limitation of the volumes of fluids used inrinsing and regeneration that has led industry to go further in thedevelopment of continuous operations.

Despite their simplicity, batch systems are still rather unsuitable forcertain problems of ion exchange, in particular when the solution to betreated has a very high level of impurities. In fact, the volume ofresins employed is not calculated in relation to the hourly flow ratebut in relation to the desired autonomy between two cycles, which leadsto the immobilization of volumes of exchanger that may be considerable.The quest for continuity of operations is a general trend in industryand in the food industry in particular. The advantages expected from itare, in addition to continuous flows, a decrease in volumes in interimstorage, most precise dimensioning of the equipment for treating theseaverage flows, and a constant quantity of products and effluents.

Numerous processes have been proposed for carrying out continuous ionexchange.

Plant has been designed in which the resin is transported hydraulicallyor pneumatically. U.S. Pat. No. 2,815,332 describes a closed-circuitsystem in which the resin advances in countercurrent to the liquid. Thisloop contains four zones isolated by valves and dedicated respectivelyfor saturation, rinsing, regeneration and rinsing. The resin advancesthrough the zones and from one compartment to the next, incountercurrent to the liquid phase, under the action of hydraulicpulses. In such a system, the saturated resin layers are removedsemi-continuously, i.e. by pulsation with a frequency of severalminutes, and are replaced with freshly regenerated resin that isintroduced on the side where the treated liquid leaves the fixationcolumn. Rapid circulation of the resin reduces the total volume of resinemployed. It is stated that it is possible, in particular, to use asingle installation instead of two operating alternately. Evidently suchsystems are very difficult to implement.

Certain authors have developed a system called SMB (Simulated MovingBed) for chromatography. Thus, U.S. Pat. No. 2,985,589 describes acontinuous SMB chromatography process, in which the chromatography resinis fixed, distributed in several compartments, but its movement issimulated by the displacement at regular intervals of the position ofthe fluid inlets and outlets. The positions of the inlets (feed andeluent) and of the outlets (extract and raffinate) then define fourzones. U.S. Pat. No. 6,375,851 describes a system with six zones, anadaptation to ion exchange of the process described previously in U.S.Pat. No. 2,985,589. The system described in document U.S. Pat. No.6,375,851 is based on an SMB, except for the regeneration stage, whichis implemented with displacement of the front. The fluid inlets andoutlets are therefore shifted, in general simultaneously, synchronouslyafter a sequence. The number of valves in these systems is prohibitive,which explains why these ionic SMB systems have never been implementedindustrially, to the benefit of the AST solution, currently the onlycontinuous ion exchange solution on the market.

Installations have also been designed in which the resin is placed incolumns arranged as a turntable or carousel. A certain number ofelementary columns corresponding to each phase of the ion exchangecycle, namely production, regeneration and the various rinsing stages,are arranged in a carousel. Such a system is described in FR-A-1546823.In this system, the columns of resins are placed on a rotating ring, therotation of which is controlled by a timer, bringing each columnsuccessively to the various treatment phases. The columns are connectedto a rotary distributor divided into as many cells as there are columns.The effluents are collected in a circular channel divided into as manysections as there are phases. Thus, each column passes successivelythrough all the stages of the cycle. U.S. Pat. No. 4,522,726 describes asystem suitable for ion exchange in which the cells containing theadsorbent are arranged in a carousel and are fed by a multi-way rotaryvalve distributing the fluids into the various compartments. The cellsare presented successively in front of the ports of this valve, which issupplied continuously. It is this last-mentioned system that is used atpresent by the company AST in devices with 20 or 30 carousel-mountedcolumns. This system has several drawbacks. On the one hand, thecarousel with the columns fitted is a complex system, as is the valve,the construction and maintenance of which are difficult. Moreover, thesequence time is determined by the smallest unit operation, each columnundergoing a single operation during one sequence. Therefore a largenumber of columns is required, to take account of the differences insequence time for each unit operation. Furthermore, as the unitoperations are not of the same duration, to operate the carousel it istherefore necessary for certain unit operations to distribute fluid to 2or 3 columns, which poses problems of hydraulic equilibrium. In fact, asit is impossible on cost grounds to fit control flowmeters on each pipegoing to each column, it is not possible to ensure that the same amountof fluid flows effectively into each column. Moreover, there is noflexibility in this system. In addition, U.S. Pat. No. 5,156,736describes a simulated moving bed in a single column, with at least twoinjection points, two collecting points and a recycling loop, for theseparation of sugars.

In all these systems, the authors envisage perfect continuity ofcirculation of all the fluids. These systems lead to a very large numberof columns, the size of the columns being defined by the smallestsequence in the series.

U.S. Pat. No. 6,280,623 describes a multi-cell system, each cellcontaining one or more stages with different solids. The principle ofcontact between the phases is the fluidized bed, in order to improvetransfer of material and efficient utilization of the stationary phase.The technique described is not chromatography.

U.S. Pat. No. 6,245,238 describes a device for chromatography bylow-pressure displacement for ion exchange, for pharmaceuticals,peptides, proteins, oligonucleotides and vaccines. The device makes itpossible to connect several columns in series (during feed injection,the columns are refilled) and in parallel (after charging, the productcontained in each column is discharged from all the columns at the sametime).

For processes applied to biomolecules, and for example ion exchange oraffinity processes, the production and regeneration stages are followedsystematically by rinsing stages. In batch processes, the stages aremost often carried out at the optimum flow rate with respect to thefixation or desorption kinetics. In continuous mode, this is not thecase, and the optimum hydraulic conditions for each stage of thesequence are therefore not maintained in the continuous processesdescribed above.

SUMMARY OF THE INVENTION

The applicant has developed a semi-continuous multicolumn sequentialprocess applied to the purification of molecules and in particularbiomolecules as defined in the present invention, which makes itpossible to obtain results and yields that are much better than existingresults, and on an industrial scale.

The processes according to the invention, as described in the claims,were elaborated on the basis of these findings.

The invention in particular provides a process for separatingbiomolecules from an aqueous solution containing said biomolecule andimpurities, having different affinities and/or interactions with thesolid support.

The invention therefore provides a process for separation on a solidsupport by multicolumn sequential selective retention for separating aproduct of interest from a solution containing said product of interest,by passing this solution over a fixed bed of chromatographic resincomprising at least three zones, means for flow of liquid being arrangedbetween adjacent zones and between the last and the first zone, saidprocess comprising several sequences, each sequence comprising at leastone stage selected from an adsorption stage, a rinsing stage, adesorption stage, implemented simultaneously or not simultaneously, eachsubsequent sequence being carried out by the downstream displacement ofthe fronts in the zones by approximately the same increment before theperiodical displacement of the introduction and withdrawal points,characterized in that the process includes a sub-sequence without feedinjection.

The invention also provides a process for separation on a solid supportby multicolumn sequential selective retention for separating a productof interest from a solution containing said product of interest, bypassing this solution over a fixed bed of chromatographic resincomprising at least three zones, means for flow of liquid being arrangedbetween adjacent zones and between the last and the first zone, saidprocess comprising several sequences, each sequence comprising thestages of adsorption, of rinsing, and of desorption, implementedsimultaneously or not, each subsequent sequence being carried out bydownstream displacement of the fronts in the zones by approximately thesame increment before the periodical displacement of the introductionand withdrawal points, characterized in that the process includes asub-sequence without feed injection.

The invention also provides a process for separating a product ofinterest from a solution containing said product of interest andimpurities, by passing this solution over a fixed bed of chromatographicresin comprising at least four zones in series, means for flow of liquidbeing arranged between adjacent zones and between the last and the firstzones, said product of interest being selectively retained by contactwith said chromatographic resin and at least one of the impurities beingretained relatively less on this chromatographic resin than said productof interest, said chromatographic resin being regenerated by the actionof a regenerating agent, characterized in that it comprises severalsequences, each sequence including at least one of the following stages:

(a) introduction of a certain volume of a rinsing solution at the inletof the first zone and approximately simultaneous withdrawal of the samevolume of a liquid diluted with said product of interest, at a pointsituated downstream of this zone;

(b) introduction of a certain volume of said feed solution at the inletof the second zone and approximately simultaneous withdrawal of the samevolume of a liquid rich in the impurity or impurities that are retainedrelatively less, at a point situated downstream of this zone;

(c) introduction of a certain volume of a rinsing solution at the inletof the third zone and approximately simultaneous withdrawal of the samevolume of a liquid diluted with regenerating agent, at a point situateddownstream of this zone;

(d) introduction of a certain volume of regenerating agent at the inletof the fourth zone and approximately simultaneous withdrawal of the samevolume of a diluted liquid, at a point situated downstream of this zone;

(e) introduction of a certain volume of an eluent at the inlet of thefifth zone and approximately simultaneous withdrawal of the same volumeof a liquid rich in said product of interest, at a point situateddownstream of this zone;

it being possible for stages (a), (b), (c), (d) and (e) to beimplemented simultaneously or not simultaneously;

each subsequent sequence being carried out by periodical downstreamdisplacement, by approximately the same volume increment, of theintroduction and withdrawal points,

and additionally comprising a stage

(f) displacement of the fronts in at least zone (b) before theperiodical displacement.

The invention also provides a process for separating a product ofinterest from a solution containing such a product of interest andimpurities, by passing this solution over a fixed bed of chromatographicresin comprising at least four zones in series, means for flow of liquidbeing arranged between adjacent zones and between the last and the firstzones, said product of interest being selectively retained by contactwith said chromatographic resin and at least one of the impurities beingretained relatively less on this chromatographic resin than said productof interest, said chromatographic resin being regenerated by the actionof a regenerating agent, characterized in that it comprises severalsequences, each sequence comprising at least one of the followingstages:

(a) introduction of a certain volume of a rinsing solution at the inletof the first zone and approximately simultaneous withdrawal of the samevolume of a liquid diluted with said product of interest, at a pointsituated downstream of this zone;

(b) introduction of a certain volume of said feed solution at the inletof the second zone and approximately simultaneous withdrawal of the samevolume of a liquid rich in the impurity or impurities that are retainedrelatively less, at a point situated downstream of this zone;

(e) introduction of a certain volume of an eluent at the inlet of thethird zone and approximately simultaneous withdrawal of the same volumeof a liquid rich in said product of interest, at a point situateddownstream of this zone;

it being possible for stages (a), (b) and (e) to be implementedsimultaneously or not simultaneously;

each subsequent sequence being carried out by the periodical downstreamdisplacement, by approximately the same volume increment, of theintroduction and withdrawal points,

and comprising moreover a stage

(f) displacement of the fronts in at least zone (b) before theperiodical displacement.

The invention also provides a process for separating a product ofinterest from a solution containing said product of interest andimpurities, by passing this solution over a fixed bed of chromatographicresin comprising at least four zones in series, means for flow of liquidbeing arranged between adjacent zones and between the last and the firstzones, said product of interest being selectively retained by contactwith said chromatographic resin and at least one of the impurities beingretained relatively less on this chromatographic resin than said productof interest, said chromatographic resin being regenerated by the actionof a regenerating agent, characterized in that it comprises severalsequences, each sequence comprising the following stages:

(a) introduction of a certain volume of a rinsing solution at the inletof the first zone and approximately simultaneous withdrawal of the samevolume of a liquid diluted with said product of interest, at a pointsituated downstream of this zone;

(b) introduction of a certain volume of said feed solution at the inletof the second zone and approximately simultaneous withdrawal of the samevolume of a liquid rich in the impurity or impurities that are retainedrelatively less, at a point situated downstream of this zone;

(c) introduction of a certain volume of a rinsing solution at the inletof the third zone and approximately simultaneous withdrawal of the samevolume of a liquid diluted with regenerating agent, at a point situateddownstream of this zone;

(d) introduction of a certain volume of regenerating agent at the inletof the fourth zone and approximately simultaneous withdrawal of the samevolume of a diluted liquid, at a point situated downstream of this zone;

(e) introduction of a certain volume of an eluent at the inlet of thefifth zone and approximately simultaneous withdrawal of the same volumeof a liquid rich in said product of interest, at a point situateddownstream of this zone;

it being possible for stages (a), (b), (c), (d) and (e) to beimplemented simultaneously or not simultaneously;

each subsequent sequence being carried out by the periodical downstreamdisplacement, by approximately the same volume increment, of theintroduction and withdrawal points,

and comprising moreover a stage

(f) displacement of the fronts in at least zone (b) before theperiodical displacement.

The invention also provides a process for separating a product ofinterest from a solution containing said product of interest andimpurities, by passing this solution over a fixed bed of chromatographicresin comprising at least four zones in series, means for flow of liquidbeing arranged between adjacent zones and between the last and the firstzones, said product of interest being selectively retained by contactwith said chromatographic resin and at least one of the impurities beingretained relatively less on this chromatographic resin than said productof interest, said chromatographic resin being regenerated by the actionof a regenerating agent, characterized in that it comprises severalsequences, each sequence comprising at least one of the followingstages:

(a) introduction of a certain volume of an equilibration solution at theinlet of the first zone and approximately simultaneous withdrawal of thesame volume of a liquid composed firstly of regenerating solution andthen of the equilibration solution, at a point situated downstream ofthis zone;

(b) introduction of a certain volume of the solution of feed to betreated containing the product of interest at the inlet of the secondzone and approximately simultaneous withdrawal of the same volume of aliquid containing the impurity or impurities that are retainedrelatively less, at a point situated downstream of this zone;

(c) introduction of a certain volume of a rinsing solution at the inletof the third zone and approximately simultaneous withdrawal of the samevolume of a liquid diluted with the impurity or impurities that areretained relatively less than the product of interest, at a pointsituated downstream of this zone;

(d) introduction of a certain volume of eluting solution at the inlet ofthe fourth zone and approximately simultaneous withdrawal of the samevolume of a liquid, containing the product of interest, at a pointsituated downstream of this zone;

(e) introduction of a certain volume of a regenerating solution at theinlet of the fifth zone and approximately simultaneous withdrawal of thesame volume of a liquid containing the impurities that are retainedmost, at a point situated downstream of this zone;

it being possible for stages (a), (b), (c), (d) and (e) to beimplemented simultaneously or not;

each subsequent sequence being carried out by the periodical downstreamdisplacement, by approximately the same volume increment, of theintroduction and withdrawal points,

and comprising moreover a stage

(f) displacement of the fronts in at least zone (c) before theperiodical displacement.

The invention also provides a process for separating a product ofinterest from a solution containing such a product of interest andimpurities, by passing this solution over a fixed bed of chromatographicresin comprising at least four zones in series, means for flow of liquidbeing arranged between adjacent zones and between the last and the firstzones, said product of interest being selectively retained by contactwith said chromatographic resin and at least one of the impurities beingretained relatively less on this chromatographic resin than said productof interest, said chromatographic resin being regenerated by the actionof a regenerating agent, characterized in that it comprises severalsequences, each sequence comprising at least one of the followingstages:

(b) introduction of a certain volume of the solution of feed to betreated containing the product of interest at the inlet of the firstzone and approximately simultaneous withdrawal of the same volume of aliquid containing the impurity or impurities that are retainedrelatively less, at a point situated downstream of this zone;

(c) introduction of a certain volume of a rinsing solution at the inletof the second zone and approximately simultaneous withdrawal of the samevolume of a liquid diluted with the impurity or impurities that areretained relatively less than the product of interest, at a pointsituated downstream of this zone;

(e) introduction of a certain volume of a regenerating solution at theinlet of the third zone and approximately simultaneous withdrawal of thesame volume of a liquid containing the impurities that are retainedmost, at a point situated downstream of this zone;

it being possible for stages (b), (c) and (e) to be implementedsimultaneously or not simultaneously;

each subsequent sequence being carried out by the periodical downstreamdisplacement, by approximately the same volume increment, of theintroduction and withdrawal points,

and comprising moreover a stage

(f) displacement of the fronts in at least zone (c) before theperiodical displacement.

According to one embodiment, stages (d) and (e) are carried out with thesame fluid, these stages then corresponding to a stage consisting of:

(d) introduction of a certain volume of regenerating agent at the inletof the fourth zone and approximately simultaneous withdrawal of the samevolume of a liquid rich in said product of interest, at a point situateddownstream of this zone;

the fourth and fifth zones then being merged into a single fourth zone.

According to one embodiment, stages (a), (b), (c) and (d) areimplemented at least partly simultaneously.

According to one embodiment, said displacement of the fronts displacesthe fronts synchronously in the different zones.

According to one embodiment, the displacement of the fronts comprisesthe following stages:

(i) creation of a circulation loop zone between the different zones,from the first zone to the fifth zone; and

(ii) circulating in said loop to displace the fronts.

According to one embodiment, the displacement of the fronts comprisesthe following stages:

(i) creation of a first displacement zone by fluid connection from theoutlet of the first zone to the inlet of the second zone and by fluidconnection from the outlet of the second zone to the inlet of the thirdzone, and downstream displacement of the inlet of the first zone toprovide the inlet of the first displacement zone and upstreamdisplacement of the outlet of the third zone to provide the outlet ofthe first displacement zone; and

creation of a second displacement zone by fluid connection from theoutlet of the third zone to the inlet of the fourth zone and by fluidconnection from the outlet of the fourth zone to the inlet of the fifthzone and fluid connection from the outlet of the fifth zone to the inletof the first zone, and downstream displacement of the inlet of the thirdzone to provide the inlet of the second displacement zone and upstreamdisplacement of the outlet of the first zone to provide the outlet ofthe second displacement zone; and

(ii) introduction of a certain volume of rinsing solution at the inletof the first displacement zone and approximately simultaneous withdrawalof the same volume of rinsing solution recovered at the outlet of thefirst displacement zone.

(iii) introduction of a certain volume of rinsing solution at the inletof the second displacement zone and approximately simultaneouswithdrawal of the same volume of rinsing solution recovered at theoutlet of the second displacement zone.

According to one embodiment, said displacement of the fronts displacesthe fronts asynchronously in the different zones.

According to one embodiment, the displacement of the fronts comprisesthe following stages:

(i) creation of a first zone of a first displacement by fluid connectionfrom the outlet of the first zone to the inlet of the second zone and byfluid connection from the outlet of the second zone to the inlet of thethird zone; and

creation of a second zone of a first displacement by fluid connectionfrom the outlet of the third zone to the inlet of the fourth zone and byfluid connection from the outlet of the fourth zone to the inlet of thefifth zone and by fluid connection from the outlet of the fifth zone tothe inlet of the first zone; and

(ii) introduction of a certain volume of said solution at the inlet ofthe first displacement zone and approximately simultaneous withdrawal ofthe same volume of a liquid diluted with regenerating agent at theoutlet from the first zone of a first displacement;

(iii) introduction of a certain volume of regenerating agent at theinlet of the second displacement zone and approximately simultaneouswithdrawal of the same volume of a liquid diluted with said product ofinterest at the outlet from the second zone of a first displacement;

(iv) creation of a first zone of a second displacement by fluidconnection from the outlet of the first zone to the inlet of the secondzone and by fluid connection from the outlet of the second zone to theinlet of the third zone, and downstream displacement of the inlet of thefirst zone to provide the inlet of the first zone of a seconddisplacement and upstream displacement of the outlet of the third zoneto provide the outlet of the first zone of a second displacement; and

creation of a second zone of a second displacement by fluid connectionfrom the outlet of the third zone to the inlet of the fourth zone and byfluid connection from the outlet of the fourth zone to the inlet of thefifth zone and fluid connection from the outlet of the fifth zone to theinlet of the first zone, and downstream displacement of the inlet of thethird zone to provide the inlet of the second displacement zone andupstream displacement of the outlet of the first zone to provide theoutlet of the second displacement zone; and

(vi) introduction of a certain volume of rinsing solution at the inletof the first zone of a second displacement and approximatelysimultaneous withdrawal of the same volume of a liquid rich in theimpurity or impurities that are retained relatively less at the outletfrom the first zone of a second displacement.

(vii) introduction of a certain volume of rinsing solution at the inletof the second zone of a second displacement and approximatelysimultaneous withdrawal of the same volume of a liquid rich in saidproduct of interest at the outlet from the second zone of a seconddisplacement.

According to one embodiment, the first, second, third, fourth and fifthzones comprise at least one column, preferably at least two columns.

According to one embodiment, stage (f) comprises a displacement of thefronts in all the zones before the periodical displacement.

According to one embodiment, the periodical displacement of theinjection points is carried out from one zone to one zone.

According to one embodiment, the periodical displacement of theinjection points is carried out from one column to one column.

According to one embodiment, the periodical displacement of theinjection points is carried out from two columns to two columns.

According to one embodiment, said zones comprise at least one column,preferably at least two columns.

According to one embodiment, the volume increment according to whichsaid points of introduction and said points of withdrawal are displacedcorresponds approximately to the volume of an entire fraction of a zoneof absorbent material.

According to one embodiment, the volume increment according to whichsaid points of introduction and said points of withdrawal are displacedcorresponds approximately to the volume of a column.

According to one embodiment, the columns are equipped with multi-wayvalves.

According to one embodiment, the periodical displacement of the stagesis synchronous.

According to one embodiment, the periodical displacement of the stagesis asynchronous.

According to one embodiment, said liquid diluted with said product ofinterest is sent at least partly to stage (b).

According to one embodiment, there is a supplementary zone, and theprocess additionally comprises a stage (g) of introduction of all orpart of the liquid diluted with said product of interest obtained instage (a) at the level of said supplementary zone, and recovery ofapproximately the same volume of rinsing solution at a point situateddownstream of this zone.

According to one embodiment, stage (b) comprises two substages (b1) and(b2), as well as an intermediate stage of adjustment of a parameter ofthe solution, in particular by altering the pH.

According to one embodiment, said liquid diluted with regenerating agentobtained in stage (c) is sent at least partly to stage (d), optionallyafter being topped up.

According to one embodiment, the recovered rinsing solutions are sent atleast partly to stages (a) and/or (c).

According to one embodiment, the chromatography is an ion exchangechromatography and the product of interest is an amino acid, preferablylysine.

According to one embodiment, the chromatography is an ion exchangechromatography and the product of interest is insulin.

According to one embodiment, the chromatography is an affinitychromatography and the product of interest is IgG.

According to one embodiment, the chromatography is an affinitychromatography and the product of interest is a protein or abiomolecule.

Preferably, the solvent (or buffer) of the rinsing solution is differentfrom the solvent (or buffer) of the desorption solution (pH,composition, eluting power etc.); preferably, the solvent (or buffer) ofthe rinsing solution is the same as the solvent (or buffer) foradsorption (pH, composition, eluting power etc.).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic representation of an SMB embodiment accordingto the prior art;

FIG. 2 is a diagrammatic representation of a column in its environmentin an installation according to one embodiment of the invention.

FIG. 3 is a diagrammatic representation of a first embodiment;

FIG. 4 is a diagrammatic representation of a second embodiment;

FIG. 5 is a diagrammatic representation of a third embodiment;

FIG. 6 is a diagrammatic representation of a fourth embodiment;

FIG. 7 illustrates an example of partial charging of a column with afeed containing for example an immunoglobulin on a stationary phasecontaining for example a protein ligand A;

FIG. 8 illustrates the charging of three columns with a third of thevolume of the column in FIG. 7;

FIG. 9 is a diagrammatic representation of an SMB embodiment accordingto the prior art;

FIGS. 10A and 10B are diagrammatic representations of a displacement ofthe fronts corresponding to a transition sequence according to theinvention according to one embodiment;

FIG. 11 is a diagrammatic representation of an asynchronous displacementof a rinsing injection line according to the invention according toanother embodiment;

FIG. 12 is a diagrammatic representation of an asynchronous displacementof the feed line with a transition sequence according to the inventionaccording to another embodiment;

FIG. 13 represents a process according to the invention with 6 lines offluids;

FIG. 14A, 14B, and 14C represent the different sequences of a processaccording to the invention implemented with 5 fluids;

FIG. 15 represents the different sequences for the separation of IgG;

FIG. 16 represents the different sequences for the separation ofinsulin.

The following symbols are used in these figures:

-   W water-   FD diluted lysine-   F aqueous lysine solution—feed solution or “feed” containing a    molecule of interest (for example lysine, insulin, human IgG)-   RAF raffinate-   RD diluted regenerating solution-   R regenerating agent-   EXT extract-   Lysine displ displacement of lysine-   Ads adsorption-   Pre ads pre-adsorption-   Water displ. displacement with water (rinsing)-   Water --   NH₃ displ. displacement with water (regeneration)-   Spare --   Wash washing-   Rege regeneration-   Wash neutral neutral washing-   Acidified water --   Waste effluent

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

According to the present invention, “biomolecules” means all moleculesisolated and purified from a biological material, including, withoutbeing limiting, amino acids, proteins, peptides, hormones, antibiotics,antibodies, enzymes, components of the cell membrane such asphospholipids etc. as well as their recombinant forms or forms labelledwith hot or cold fluorescent labels well known to a person skilled inthe art.

According to the invention, the molecule of interest can be the moleculethat is retained most by the stationary phase but also the moleculeretained least by the stationary phase. As an example it may bementioned that when the adsorption process according to the invention isused for the separation of endotoxins, the toxins are the moleculesretained most by the stationary phase and the molecule of interest isretained least.

According to the present invention, by “adsorption stage” is meant astage in the course of which feed containing the molecule to beseparated is injected; one or more of the products contained in the feedwill then become attached to the solid phase. This stage corresponds tocharging of the phase.

According to the present invention, by “desorption stage” is meant astage in the course of which the product or products that are fixed onthe solid support pass into the liquid phase. An adsorption processtherefore naturally comprises at least one adsorption stage and at leastone desorption stage.

According to the present invention, by “rinsing stage” is meant a stagebetween an adsorption stage and a desorption or reversal stagepermitting renewal of the liquid phase contained in the column orcolumns. The designation “washing stage” may also correspond to thisstage.

The process of the invention permits a better optimization of thesequences of feed of the aqueous solution to be treated and rinsing ofthe separated products; this leads to a reduction in the volumes usedand to less production of by-products.

Moreover, the process according to the invention also offers one or moreof the following advantages:

-   -   mechanical design that makes it possible to avoid moving parts,        since the columns are fixed, and the carousel is no longer        required. The columns are compact and it is possible to use        multi-way valves on each column.    -   simplified maintenance, since a column can be separated from the        cycle without having to stop the process, for changing the        chromatographic resin or support, general maintenance, etc. The        maintenance requirements are close to those of a discontinuous        process, which are known to be relatively low.    -   process control is simple, since it is sufficient to change the        parameters of the automatic system in order to modify the        process and adjust the process zones (no mechanical component        has to be changed). Furthermore, it is possible to use a        flowmeter on each column, for each process stage.    -   expansion of capacity is easy to implement, simply by adding        columns to the existing columns and by modifying the process        parameters in order to modify the zones.    -   the required floor space is also less than with a system of the        prior art using a carousel.

These advantages, as well as others, will be explained as appropriate inthe description that follows.

For FIGS. 1, 3 to 6, the description is given with reference to aspecific amino acid, lysine, but it is understood that the processaccording to the invention applies to all the amino acids and moregenerally to all the biomolecules according to the invention, andapplies to any type of chromatography well known to a person skilled inthe art.

It will be recalled that the biomolecule is regarded, for ion exchange,as the extract (X) as it is exchanged more and that the impurities thatare exchanged less are regarded as a raffinate (R). In the invention,the purity in the extract is for example greater than 95%, preferablygreater than 98%, advantageously greater than 99%. The same conventionswill be used as for the other examples in the present application, theterm “more retained” or “less retained” being used when there is no ionexchange.

The ion exchange resins are conventional, as are the rinsing solutionsand regenerating solutions. For example, the resins according to theinvention can be selected from the affinity, ionic, normal or reversedphase resins, with hydrophobic interaction, size-exclusion, etc. Fromthis standpoint, the invention does not differ from the prior art forion exchange. It is possible to use weak or strong, anionic or cationicresins, depending on the case to be treated.

It is also possible to use several systems in series; in particular ademineralization can be carried out using a first system with cationicresins followed by a second system with anionic resins.

It should be noted that when the biomolecule is an amino acid such aslysine, the aqueous solution can be the culture broth obtained byfermentation producing said amino acid. Water is then used as eluent inthe invention. It is in fact possible to use a culture broth as startingproduct for any type of separation, by adapting the resins in thecolumns.

Said aqueous solution containing the biomolecule can be obtained byclarification of the culture broth, and said clarification can comprisecentrifugation and/or membrane filtration, preferably membranefiltration.

The present invention is illustrated as an example by the followingdescription of three embodiments, referring to FIGS. 3 to 5 of theattached drawings, whereas FIG. 1 describes an embodiment according tothe prior art. Lysine is taken as a representative example of an aminoacid, itself representative of a substance to be purified in an ionexchange chromatography process, the latter being representative of ageneric chromatography process to which the invention applies.

Referring to FIG. 1, the installation comprises eight columns 1, 2, 3,4, 5, 6, 7 and 8 packed with ion exchange resin. The operation of thisinstallation of the SMB type according to the prior art is explainedbelow. It has to be borne in mind that the displacement stages go fromleft to right, which in fact corresponds to displacement of the columnsfrom right to left.

Stage (a) comprises the steps for introducing a certain volume of waterW into the inlet of column 1 and approximately simultaneouslywithdrawing the same volume of diluted lysine from the outlet of column2, with columns 1 and 2 forming the first zone. During introduction ofwater, right at the start of the stage, it should be borne in mind thatowing to the displacement, column 1 is therefore in fact column 2 justpreviously (before the displacement). Column 2 just previously is filledwith the diluted lysine solution (that which has not been fixed by ionexchange). The front of pure water therefore moves downwards, and column1 therefore passes from a “diluted lysine” state (it being understoodthat there is exchanged lysine in this column) to a “water” state (withexchanged lysine in this column). Column 2, which was column 3 justpreviously, passes from a “lysine” state (with fixed exchanged lysine)to a “diluted lysine” state (with fixed exchanged lysine), and lysinesolution to be purified is recovered in the diluted state at the bottomof column 2, relatively concentrated at first, then more and moredilute. In fact no more exchange sites are available in this column andtherefore the dilute solution is simply eluted along this column; thisis a displacement of lysine solution. This recovered dilute solution istypically returned to the starting tank of the solution to be purified,or else directly to the head of column 3.

Stage (b) comprises the steps for introduction of a certain volume ofsaid aqueous solution into the inlet of column 3 and approximatelysimultaneous withdrawal of the same volume of a liquid rich in raffinatefrom the outlet of column 4. Column 3 is fed with lysine solution, butthis column corresponds to column 4 immediately before, therefore to acolumn already partially exchanged with lysine, and also containingdiluted lysine that has not been exchanged. Similarly, column 4corresponds to column 5, but immediately previously and therefore acolumn with water. Column 4 is a preadsorption column since it receivesspent lysine solution from column 3. In column 3, the lysine solution isinjected and a saturation front advances in column 3, whereas the frontof the lysine solution to be purified and which is exchanged on thesites advances in column 4 (preadsorption column). At the outlet ofcolumn 4, the raffinate is recovered, i.e. the elements of the solutionto be treated that have not been exchanged with the resin, starting froma very dilute solution to a solution more concentrated with raffinate.

Stage (c) comprises the steps for introduction of a certain volume ofwater at the inlet of column 5 and approximately simultaneous withdrawalof the same volume of a liquid diluted with regenerating agent from theoutlet of column 6. Column 5 is fed with water, whereas diluteregenerating agent leaves from column 6. In fact, column 6 is column 7just before the displacement and therefore just after the displacementit receives what leaves column 5, namely water with a small amount ofregenerating agent. What leaves column 6 is therefore diluteregenerating agent.

Stage (d) comprises the steps for introduction of a certain volume ofregenerating agent into the inlet of column 7 and approximatelysimultaneous withdrawal of the same volume of a liquid rich in the aminoacid from the outlet of column 8. Column 7 is fed with regeneratingagent, and is connected to column 8. This column 8 is column 1 justbefore the displacement and therefore just after the displacement column8 is fed with regenerating agent, with the result that the regeneratingagent front agent advances in column 8, and the extract is thenrecovered at the bottom of column 8, dilute at first and then more andmore concentrated and when the amount recovered begins to decrease, thestages are displaced.

At the end of a given sequence N, at the head of column 1 there istherefore water and exchanged lysine fixed on the resin. At the head ofcolumn 2 there is exchanged lysine, water and a residue of lysinesolution to be purified. At the head of column 3 there is completelyexchanged lysine, with lysine solution to be purified. At the head ofcolumn 4, there is partially exchanged lysine (not all the sites areexchanged) and raffinate (portion of the lysine solution to be purifiedthat has not been fixed) and a residue of lysine solution to bepurified. At the head of column 5, there are rinsing water and the sitesof the resin that are ready to be exchanged. At the head of column 6,there is rinsing water diluted with regenerating agent, with the sitesbeing regenerated. At the head of column 7, there are the fullyregenerated sites (ready to exchange lysine) and regenerating solution.At the head of column 8, there are partially regenerated sites anddiluted regenerating solution and extract (partially).

Therefore right at the start of the next sequence N+1, and withreference to a scheme in which the columns are displaced towards theleft by displacement of the injection and withdrawal points towards theright, the positions are then as follows. Water is sent to column 2,which at its head has exchanged lysine, water and a residue of lysinesolution to be purified. At the head of column 3 there is fullyexchanged lysine, with lysine solution to be purified, which will thenreceive what leaves column 2. At the head of column 4, there ispartially exchanged lysine (not all the sites are exchanged) andraffinate (portion of the lysine solution to be purified that has notbeen fixed) and a residue of lysine solution to be purified, and thiscolumn then receives the lysine solution to be purified. At the head ofcolumn 5, there are the rinsing water and the sites of the resin readyto be exchanged, and it receives what leaves column 4, namely raffinateand depleted lysine solution which will be exchanged on thispreadsorption column. At the head of column 6, rinsing water is fed to acolumn having regenerated sites, and containing dilute solution ofregenerating agent. Column 7, which also has fully regenerated sites(ready to exchange lysine), then receives water from column 6 anddiluted regenerating solution which is withdrawn at the bottom. At thehead of column 8, there are partially regenerated sites and dilutedregenerating solution, and a solution of regenerating agent is supplied.

The flows that will arrive at the head of the columns will therefore beas follows. At the head of column 2 the solution to be purified arriveson a column already having partially exchanged lysine at its head. Thereis therefore a breakdown of the fronts and a gradient that is notrespected. At the head of column 8 the regenerating solution arrives ona column partially regenerated with dilute regenerating agent, andtherefore there is, locally, mixing of pure regenerating agent withimpure regenerating agent. Once again, the gradient is not respected.

We therefore see that the conventional SMB system, applied in the fieldof ion exchange resins, although having the advantage of beingcontinuous, is not free from problems, which are especially marked inregeneration.

Owing to a stage of displacement of the fronts in the zones before theperiodical displacement, the invention makes it possible to overcomethis problem and respect the gradients. The stage of displacement of thefronts is carried out typically for one column. The description thatfollows makes reference to three embodiments. Displacement of the frontsis carried out by injection of a selected fluid into the columns, eitherthe existing fluids by circulation in a closed loop, by injection ofwater (the rinsing liquid) or by injection of feed and of regeneratingagent followed by injection of water.

In FIGS. 3, 4 and 5, the installation is identical in terms of columns,only differing in the operating mode of the columns, and the possiblepresence of intermediate tanks (not shown).

The installation comprises columns (1, 2, 3, 4, 5, 6, 7 and 8) packedwith the same amount of an ion exchange resin, for example strongcationic resins of the polystyrene type (for example with 4-10% ofdivinyl benzene, advantageously approximately 8%), such as the resinsDIAONS SK1 B from Mitsubishi, DOWEX 600 B from Dow, or FPC 11-Na fromRohm & Haas.

These columns are arranged in series, each comprising an inlet and anoutlet. In general, as will be seen later, each inlet can receive theaqueous solution to be treated, the regenerating solution, water,recovered water, acidified lysine, and ammonia. In general, as will beseen later, each outlet can produce the diluted lysine, raffinate,extract, recovered water, dilute ammonia, and lysine (extract). Eachcolumn is, moreover, connected to the column upstream and downstream.

This principle is shown in FIG. 2. As shown in FIG. 2, the valves can bemulti-way valves, in particular 6-way. These multi-way valves are knownper se, and are operated conventionally by an electric motor.Advantageously, the valves are actuated to turn by an increment eachtime in an operating mode. For certain operating modes, the valves canbe actuated to turn by several sectors, for example when a column is tobe isolated to carry out a specific sequence on said column.

With reference to the first embodiment shown in FIG. 3, after sequenceN, identified as sequence 1.1 in the diagram, there is the arrangementdescribed above with reference to the SMB system.

Then a displacement of the fronts is carried out during sequence 1.2.This displacement is obtained by putting the columns in a loop and bycirculation.

This displacement is carried out by an increment of one column, bycirculating the fluids in the loop. The volume displaced corresponds tothe volume of a column.

Thus, at the start of sequence N+1 the following columns are obtained.Column 4 therefore has, at its head, partially exchanged sites and interms of fluid, what was at the head of column 3 (the sites alreadyexchanged do not change), therefore lysine solution to be purified(therefore a composition approximately identical to what the column willreceive next). Column 8 then has partially regenerated sites andsolution of regenerating agent (therefore a composition approximatelyidentical to what the column will receive next). Thus, the columns arefed with a constant gradient, since the concentrations do not change atthe head of these columns. This is also true for the other columns.Column 6 has, at its head, what was at the head of column 5, namelywater, and will also receive water. Column 2 has, at its head, what wasat the head of column 1, namely water, and will also receive water.

Referring to the second embodiment shown in FIG. 3, after sequence N,identified as sequence 1.1 in the diagram, there is the arrangementdescribed above with reference to the SMB system.

A displacement of the fronts is then carried out during sequence 1.2.This displacement is obtained by circulating two displacement zones. Thefirst displacement zone is the zone comprising columns 2, 3, 4 and 5.The second displacement zone is the zone comprising columns 6, 7, 8 and1.

This displacement is carried out by an increment of one column, byinjection of water at the head of the displacement zones. The volumedisplaced corresponds to the volume of a column.

Therefore at the start of the sequence N+1 the columns are obtained asin the first embodiment. The difference comes from the displacement of acolumn volume of water. In fact, between the two parts of theproduction/regeneration process, there is a buffer of water to preventcontamination of the different species. This buffer of water is simplydisplaced in the first embodiment, whereas it is replaced in the secondembodiment. In the second embodiment, we then recover, at the bottom ofcolumns 1 and 5, the volume of water in a column, therefore recoveredwater (Rec W). This recovered water is sent to an intermediate tank, andcan be used for feeding the columns with rinsing water. This water canalso be used partly with fresh water for rinsing. The other sequencesare those as in the first embodiment.

In the first and second embodiments, the displacement of the fronts issynchronous, since all the fronts move at the same time by a volumeincrement. The entering fronts are displaced synchronously with theleaving fronts.

In the first and second embodiments, the sub-sequence without feedinjection corresponds to sub-sequence 1.2 (or 2.2, depending on thesequence considered).

Referring to the third embodiment shown in FIG. 5, after sequence N,identified as sequence 1.1 in the diagram, the arrangement is asdescribed above with reference to the SMB system.

Then a first displacement of the fronts is carried out during sequence1.2. This first displacement is obtained by circulating two zones offirst displacement. The first zone of first displacement is the zonecomprising columns 3, 4, 5 and 6. The second zone of first displacementis the zone comprising columns 7, 8, 1 and 2. Lysine solution isinjected into column 3, which causes a first displacement in the firstzone. At the head of column 4 there is then lysine solution to bepurified. The contents of column 6 are recovered at the bottom; it isdilute regenerating agent. Regenerating agent is injected into column 7,which causes a first displacement in the second zone. At the head ofcolumn 8 there is then solution of regenerating agent.

A second displacement of the fronts is then carried out during sequence1.3. This second displacement is obtained by circulating two zones ofsecond displacement. The first zone of second displacement is the zonecomprising columns 2, 3, 4 and 5. The second zone of second displacementis the zone comprising columns 6, 7, 8 and 1. This time water isinjected into columns 2 and 6. This then causes a second displacement ofthe fronts. The composition of the head of column 3 is now at the headof column 4; once again it is lysine solution to be purified. From thisstandpoint, the composition of the head of column 4 has not changed inthe course of this second displacement. Similarly, at column 8 we obtainthe composition of the head of column 7, namely regenerating agent.Again from this standpoint, the composition of the head of column 8 hasnot changed. What has changed in the course of this second displacementare the compositions in columns 5 and 1, since raffinate was obtained atthe bottom of column 5 and extract at the bottom of column 1. Duringthis second displacement, the “buffer” of water between the two parts ofthe production/regeneration process was reconstituted to preventcontamination of the different species.

In the third embodiment, the displacement of the fronts is asynchronous,since all the entering fronts are not displaced synchronously with theleaving fronts. In the example, the entering fronts are displaced first,then the leaving fronts are displaced, but it is also possible to do thereverse.

In the third embodiment, the sub-sequence without feed injectioncorresponds to sub-sequence 1.3 (or 2.3, depending on the sequenceconsidered).

The number of columns in a displacement zone or in a zone correspondingto the zones (a), (b), (c) and (d) is not necessarily constant. A changein the number of columns in each zone can be beneficial for maximumutilization of each column. As an example, it is possible to have afirst set of columns in elution (displacement columns), the number ofwhich is constant, whereas the zones of production and of regenerationhave variable lengths, for example two columns in production and onecolumn in regeneration, then one column in production and two columns inregeneration. As another example, if a set of M columns is considered,there can be a complete sequence (set of all the sequences (a), (b),(c), (d) and of displacement) on M−1 or M−2 columns or M−m columns. Itis then possible to isolate one, two or m columns, for example formaintenance, on the bed of resin or on the valve and pipe assembliesattached to said column.

In fact, the process according to the invention makes it possible toimplement a selected stage on a selected column, independently of theother columns, if need be. This is impossible with the continuousprocesses of the prior art. For example, as stated, it is possible toisolate a column. It is also possible, within a given sequence, tochange the feed of a column. When a column receives water, recoveredwater can be used in a first phase, and then fresh water can be sent tothis column, which makes it possible to optimize the water consumption.It is also possible to feed a column with variable feeds or variableregenerating solutions. Relative to the processes according to the priorart, it is possible to exercise better control on the rinsing andproduction flows. In particular, these processes of the prior artenvisage continuous dilution of the feed with rinsing water. This leadsinter alia to an increase in the speed of passage through the adsorptionzone (ion exchange). The optimum hydraulic conditions for each stage ofthe sequence are therefore not respected in the continuous processes ofthe prior art. The invention makes it possible to benefit most from theoptimum hydraulic conditions, by managing each stage optimally, sincestages (a), (b), (c) and (d) are not necessarily of the same duration.The flows are therefore optimized in each column. Moreover, it ispossible to use flowmeters on each column, since the number of columnsis reduced relative to a carousel with 20 or 30 columns as in the caseof the prior art of AST. With a flowmeter at the head of each column, itbecomes possible to optimize the hydraulic conditions in the column inquestion.

In the case of lysine, the process can be optimized to obtain optimizedcolumn feed fluids, by topping up the fluids leaving the columns forreuse in the next columns. Thus, during sequence 1.1 in the diagrams,the following procedure can be followed. The solution leaving column 3,which contains unfixed lysine and exchanged ammonium ions, isadvantageously reacidified with sulphuric acid, to have a neutral mediumduring feed of column 4. Ammonium sulphate is then obtained in theraffinate. The dilute lysine solution leaving column 2 can be sent tothe head of column 3 and/or can be combined with the flow leaving thiscolumn and the neutralizing acid. The solution leaving column 6, whichcontains dilute regenerating agent, can be topped up with ammonia to bereturned to the head of column 7 as regenerating agent. An extract isthen obtained which is lysine and a raffinate which contains ammoniumsulphate.

It is also possible to have other displacement columns, in particular asupplementary displacement column in stage (b), and thus have asupplementary stage of production of recovered water. This embodiment isshown in FIG. 6, which comprises the use of 9 columns. Columns 1 and 2and 3 correspond to columns 1 and 2 and 3 in FIGS. 3, 4 and 5. Columns 4and 5 correspond to column 4 in FIGS. 3, 4, 5. Column 6 is new relativeto the embodiments of FIGS. 3, 4 and 5. Column 7 corresponds to columns5 and 6 in FIGS. 3, 4, 5. Columns 8 and 9 correspond to columns 7 and 8in FIGS. 3, 4 and 5. The operating mode is identical to that for theembodiments of FIGS. 3, 4 and 5. Circulation is applied identicallyhere. Similarly, it is possible to define two displacement zonesaccording to the second embodiment as follows: the first displacementzone comprises columns 2 to 7 and the second displacement zone comprisescolumns 8, 9 and 1. It is also possible to define zones of first andsecond displacements according to the third embodiment. The first andsecond zones of first displacement comprise columns 3 to 7 on the onehand and 8 to 2 on the other hand. The first and second zones of seconddisplacement comprise columns 2 to 7 on the one hand and 8, 9 and 1 onthe other hand. In the embodiment of FIG. 6 the flow leaving column 3feeds column 4 directly, but it would also be possible to reacidify itto neutralize the effluent. In the embodiment of FIG. 6 (just as for theother embodiments), it is possible to adjust the pH of the differentfractions that are sent to the columns. For example, it is possible toensure that the pH of the fraction that has been in contact with themost charged column (here 3) is below 2 before it is sent to the nextcolumn. By changing the pH, it is also possible to vary the type oflysine fixed preferentially on the resin (monovalent or non-monovalentlysine).

The invention also applies to any type of separation by chromatographyon any type of product. In particular, the process according to theinvention can use 5 inlet fluids (or more):

-   -   Feed Fluid or Feed: this liquid contains the feed to be treated        and its pH buffer composition, salinity making it possible, by        injecting this fluid in a column, to adsorb the molecule of        interest on the stationary phase. At the end of the feeding        stage, the column contains a stationary phase on which the        component of interest is adsorbed, whereas the liquid phase        located in the column is constituted by the diluted feed fluid        (FD, Feed Diluted).    -   Rinsing fluid, with salinity and pH identical to the feed fluid,        but not containing feed to be treated. This stage renews the        liquid phase of the column and makes it possible to remove the        compounds of the feed that are not retained by the stationary        phase. In the case of lysine, for example, water or an aqueous        acid is used.    -   Elution of the desired species: a fluid of a nature modifying        the nature of the interactions between the target molecule and        the stationary phase makes it possible for the target molecule        to be desorbed from the stationary phase, the target molecule        then being collected from the outgoing liquid.    -   Regeneration: after elution, impurities may remain strongly        adsorbed on the stationary phase, and may have an adverse effect        on its stability or hygienic character. A fluid containing        additives such as soda or urea can thus be used. In the case of        lysine, described previously, the stages of elution of the        desired species and of regeneration are implemented        simultaneously, the regenerating agent (ammonia) exchanging the        sites of the resin to release the lysine.    -   Injection of a solvent corresponding to the rinsing fluid used        after feeding makes it possible to clear the column of the        regenerating solvent or solvents before carrying out the next        feed. A buffer is thus maintained between feeding and the end of        regeneration; this is called equilibration.

The invention offers a process that makes it possible to implement thefollowing 5 stages:

-   -   Stage a: called equilibration, in the course of which the        equilibration solution is injected into at least one column of        the system to clear it of the regenerating solvent that it        contains. Downstream of the equilibration zone, the fluid        withdrawn will initially be composed predominantly of the        regenerating solution, then predominantly of the equilibration        solution.    -   Stage b: called feeding, in the course of which the solution of        feed to be treated is injected. The molecule of interest is then        fixed on the chromatographic support with other impurities. At        the outlet from this zone, downstream of the injection point,        the fluid withdrawn then contains the impurities that are        retained least.    -   Stage c: called washing, in the course of which washing of the        column is carried out, replacing the liquid phase in particular        containing impurities not retained by the washing solvent.    -   Stage d: called elution, in the course of which a solution is        injected, modifying the interactions of the molecule of interest        with the chromatographic support, thus permitting the molecule        of interest to be eluted. At the outlet from the elution zone,        the fluid withdrawn will first contain the washing solution,        then a solution rich in the product of interest.    -   Stage e: called regeneration, in the course of which a        regenerating solution is injected, making it possible to detach        the impurities that are very strongly adsorbed on the support.

In the invention, the periodical displacement of the injection pointscan be carried out from one column to one column; thus, each column (orzone) can be managed independently. This variant differs in particularfrom the carousel systems of the AST type, since in these systems allthe columns are necessarily switched together. The invention makes itpossible to operate each column (or zone) independently; in particularthe displacement of the injection/withdrawal points can be synchronous,asynchronous, and similarly the stage of displacement of the fronts canbe synchronous or asynchronous, and can be applied column by column (orzone by zone). It is also possible to operate with one column (or zone)for one operation or with several columns (or zones) for otheroperations; thus it is possible to displace the injection/withdrawalpoints and the fronts of one column for one given zone and of two orseveral columns for another given zone.

The invention also applies in the case of a process using 5 fluids ormore, as stated above, and is applicable to biomolecules or othermolecules of high added value, such as medicaments. In the case ofbiomolecules, in general there is no interest in the outgoing fluids,which do not contain the molecule of interest, as the cost of thebiomolecule means that optimized stages are unnecessary. In the case ofa biomolecule, in contrast, it is essential to recover the maximumamount of said biomolecule. This principle is illustrated in particularin the following description.

In the chromatographic adsorption processes, the rate at which themolecule of interest fixes to the stationary phase is slow. To theextent that a column of volume V is fed continuously, the product leavesthe column whereas the stationary phase is not completely saturated bythe target molecule. This limited adsorption is illustrated in FIG. 7.

In FIG. 7, the upper and lower graphs show the concentration of themolecule of interest in the liquid phase and in the solid phase,respectively. The feed fluid is injected on the left of the column andit appears that the fluid leaving the column contains a non-zeroconcentration of target molecule. To continue the feeding in this waywould cause a loss of the target product, which is economicallyunacceptable. Therefore feed in fact stops whereas the product has leftthe column before complete adsorption has been carried out. The stagesof rinsing, elution, regeneration and equilibration then begin fordesorption of a target molecule adsorbed on all or part of thechromatographic resin or support. It should be noted that these stagesconsume an amount of fluids that depends mainly on the volume of thecolumn and not on the quantity of target product to be desorbed; it istherefore beneficial to have a column that is charged as much aspossible, from the standpoint of consumption of the different fluids.

If the case in FIG. 7 is now considered, but assuming that injection hasbeen carried out on three columns of volumes V/3, FIG. 8 is thenobtained.

FIG. 8 shows that under partial adsorption conditions identical to FIG.7, for the left-most column the stationary phase is completelysaturated. In the invention, the feed is no longer applied on the firstcolumn, but only on the partially saturated column 2. The subsequentstages of rinsing, elution, regeneration and equilibration are thenapplied to column 1, which is the outlet of the feed zone. In contrastto the case presented in FIG. 7, in which either the column rinsed,eluted etc., is partially charged with target molecule, the casepresented in FIG. 8 shows that column 1 is almost completely charged.The different solvents for rinsing, elution, regeneration andequilibration therefore allow more product to be desorbed in the case inFIG. 8 than in the case in FIG. 7. The consumption of eluent relative tothe amount of target molecule produced is thus reduced significantly.

Applying this principle, a synchronous, periodical and non-sequencedmulticolumn system of the AST type can be used as shown in FIG. 9. FIG.9 shows that the process involves different fluid inlets. Between eachof these inlets, it is possible to define a zone: for example betweenthe feed inlet (Feed) and the inlet of the equilibration fluid, the feedzone is defined. Between the rinsing solution (Wash) and the feed(Feed), the rinsing zone is defined, etc. These zones correspond to thefirst, second, third, fourth and fifth zones of the process according tothe invention (in the case of amino acid and ion exchange resin, thefourth and fifth zones are merged and the eluent and the regeneratingagent are the same fluid). The periodical displacement is also shown inFIG. 9. This reiterates the principle shown in FIG. 1 (whichcorresponded to the case with four fluids).

If column No. 9, at the end of the cycle is considered, it is in thefeed configuration. After switching, it is located just before theoutlet of the washing stage. Consequently, what leaves this column justafter the switching of the lines still contains the feed, thereforecontaining the molecule of interest, unpurified, which in fact becomesdiluted and is lost.

According to the invention, to avoid losing this portion of the feed anddiluting it, a displacement of the fronts is applied, as was done forthe case of the amino acid described above. The sequence is then asdescribed in FIG. 10, starting from the position in (1). Instead ofdirectly switching the feed line (Feed) and the effluent line (Waste)that precedes it, a transition stage for column No. 9 is left. In thisexample the other lines are fixed. Passing on to position (2), whichcomprises the fluid connection of the two zones, columns 8 and 9 beingin fluid connection, the other columns remaining unchanged. The rinsingsolution (identified as Wash) in white propels the feed (and thereforethe product of interest) towards column 10, which is identified byposition (3). Column 9 can then be described as “in zone transition”. Byway of transition or at the end of displacement of the fronts, position(4) is reached. The liquid contained in column 9 now contains hardly anyproduct of interest, and the feed line (Feed) and the effluent line(Waste) which precedes it can therefore reappear; this is identified atposition (5). It is then possible to proceed to the switching of theother lines.

In this case, this stage, or sub-sequence of displacement of the fronts,makes it possible to avoid losing the liquid contents of column 9, whichin this case contain the product of interest. With an affinity process,it is conventional to consider that 5 to 20 volumes of column aregenerally necessary for saturating a column. As the porosity of thechromatographic bed is approximately equal to 0.8, the amount of liquidcontained in a column is therefore 80% of the volume of the column.Avoiding the loss of an amount of feed corresponding to 0.8 columnvolume out of 5 to 20 volumes introduced will therefore make it possibleto improve the yield significantly.

This transition stage applied this time to the displacement of the lineof regenerating solvent makes it possible to economize 0.8 column volumeout of 3 to 5 column volumes injected in most cases. It can thereforealso reduce the consumption of certain regeneration solvents.

In the present invention, the feed line as well as each fluid inlet linecan in fact move freely relative to the others with the possibility ofhaving a sub-sequence of displacement of the fronts. In fact, eachdisplacement of the fronts can be carried out line by line,synchronously or asynchronously, and also line by line. All combinationscan be envisaged, it being understood that the displacement of thefronts comprises at least the displacement of the feed front, preferablyalso the displacement of the regenerating agent front. This displacementof the fronts, at least for a column that is in the feed phase, isimpossible with the systems according to the prior art.

It is also possible to carry out a displacement of the fronts by anincrement of one column, as well as by an increment less than or greaterthan one column.

A particular embodiment involves the omission of one inlet line and ofone effluent line. This case is shown in FIG. 11. In this figure, thestarting point is considered to be position (1), with lines of eluent,rinsing (Wash) and feed (Feed), the extract and effluent lines beingmarked “biomolecule” and “Waste”, without being limited to theproduction of a biomolecule as the process is applicable to any type ofproduct. In the case of FIG. 10, the periodical displacement isasynchronous. Let us consider the case where the line for feed ofrinsing solution (Wash) is displaced to 30% of the cycle time; in thiscase the column in transition during the rinsing is defined asnon-existent. Feed and Eluent are displaced to the cycle end. At 30% ofthe cycle, a displacement command is given to the line of rinsingsolution. The rinsing line therefore passes from the inlet of column 2to the inlet of column 3, being superposed on the feed line. In thespecific case under consideration, a superposition of inlet lines isundesirable, and it is defined that in such a case it is the linedownstream that takes precedence. Position (2) is reached. Symbolically,it is possible to consider a situation shown diagrammatically atposition (3).

Similarly, if three lines are superposed, it is the line downstream thattakes precedence.

The case in FIG. 12 is another particular case of periodicalasynchronous displacement. In position (1), it is assumed that at thestart of the cycle, the line of rinsing solution is superposed on thefeed line. Applying the principle of the superposition of lines, it isthe feed line that takes precedence. If the feed line is displacedasynchronously, before the line for rinsing solution, with a transitiontime (for the displacement of the fronts), position (2) is then reached.Once the transition or displacement of the fronts is carried out, inposition (3) is again reached and the displacement of the feed line iseffective.

The use of an asynchronous mode makes it possible to reduce the totalnumber of columns by causing several zones to coexist in the same columnduring a given cycle.

FIG. 13 is an example of a process according to the invention withseveral lines of fluids, numbering 6 here. The case shown in FIG. 13represents the separation of an antibiotic. In this embodiment, columns1 and 2 are supplied with water as rinsing solution, and a dilutesolution of the product of interest is obtained at the outlet. The feedis supplied to adsorption column 3 whereas the preadsorption columndownstream is supplied by the outlet of column 3 (which can be combinedwith the outlet of the washing columns 1, dilute feed solution) and aneffluent is obtained at the outlet from the preadsorption column(raffinate). Acidified water is used as equilibration solution incolumns 5 and 6, for carrying out a neutral rinsing (the columnsoriginating from a basic regeneration). The effluent leaving the rinsedcolumns 5 and 6 is combined with regeneration components, such asacetone and soda. Column 7 is then regenerated, and an effluent isobtained at the column outlet. Column 8 is submitted to washing withwater (this stage can be omitted), and sodium carbonate is added to theeffluent. This solution is an eluting solution, which is sent to columns9 and 10, the effluent from which is the extract. This extract suppliesthe product of interest and the water that is separated can be recycledto the process (shown here returned for the washing of columns 1 and 2).

Whereas the process described in FIG. 9 shows the implementation ofthese stages according to a periodical, non-sequential process in whicha displacement of the lines for injection of the different solutions iscarried out synchronously and non-sequentially, FIG. 14 shows a processin which the displacements are asynchronous and sequential. FIG. 14therefore shows an example of implementation of stages (a) to (e)according to the invention on a system with 6 columns where the cycle isbroken down into 5 sub-sequences corresponding to the displacements ofcertain lines for injection at different moments of the sequence.

At the start of the first sequence the situation is as follows:

-   -   the line for injection of the equilibration solution is at        column 1    -   the lines for injection of the regenerating solution and eluting        solution are at column 2. As explained previously, in the case        of a superposition of lines, it is the line downstream that        prevails, in this case the line for injection of eluting        solution.    -   the line for injection of the washing solution is at column 3    -   the line for injection of the solution of feed to be treated is        at column 4.

This configuration corresponds to sub-sequence 1.1 which lasts from t=0to for example t=0.24*Δt.

At the end of sub-sequence 1.1, the elution line for example isdisplaced by one column. Sub-sequence 1.2 begins, the configuration isas follows:

-   -   the line for injection of the equilibration solution is at        column 1.    -   the line for injection of the regenerating solution is at column        2.    -   the line for injection of the eluting solution is superposed on        the injection of washing solution at column 3. As the line        downstream takes precedence, it is the washing solution that is        injected at column 3.    -   the line for injection of the solution of feed to be treated is        at column 4.

Sub-sequence 1.2 lasts for example from t=0.24*Δt to t=0.36*Δt.

At the end of sub-sequence 1.2, the washing line for example isdisplaced. Sub-sequence 3 begins, the configuration is as follows.

-   -   the line for injection of the equilibration solution is at        column 1.    -   the line for injection of the regenerating solution is at column        2.    -   the line for injection of the eluting solution is at column 3.    -   the line for injection of the washing solution is superposed on        the line for injection of the solution of feed to be treated at        column 4. As the feed is downstream of the washing, it is the        injection of feed that prevails. It is therefore the solution of        feed to be treated that is injected at column 4.

Sub-sequence 1.3 lasts for example from t=0.36*Δt to t=0.60*Δt.

At the end of sub-sequence 1.3, the lines of equilibration and ofregeneration are displaced simultaneously. Sub-sequence 1.4 begins, theconfiguration is as follows.

-   -   the line for injection of the equilibration solution is at        column 2    -   the line for injection of the regenerating solution is        superposed on the line for injection of the eluting solution at        column 3. The line downstream that prevails in this case is the        injection of the eluting solution.    -   the line for injection of the washing solution is superposed on        the line for injection of the solution of feed to be treated at        column 4. As the feed is downstream of the washing, it is the        injection of feed that prevails. It is therefore the solution of        feed to be treated that is injected at column 4.

Sub-sequence 1.4 lasts for example from t=0.60*Δt to t=0.76*Δt.

At the end of sub-sequence 1.4, a transition is carried outcorresponding to the displacement of the product of interest containedin the liquid phase of column 4 to column 5. To do this, injection ofthe solution of feed to be treated is stopped, there is therefore atransition from feeding stage (b) corresponding to the displacement ofthe fronts of concentrations of the liquid phase of the unretainedimpurities and of the product of interest by stage (c). The transitionis therefore a stoppage of injection of the feed to be treatedcorresponding to the displacement of the fronts induced by stage (c).

Sub-sequence 1.5 begins, the configuration is as follows.

-   -   the line for injection of the equilibration solution is at        column 2    -   the line for injection of the regenerating solution is        superposed on the line for injection of the eluting solution at        column 3. The line downstream that prevails in this case is the        injection of the eluting solution.    -   the line for injection of the washing solution is at column 4.

Sub-sequence 1.5 lasts for example from t=0.76*Δt to t=Δt.

At the end of sub-sequence 1.5 the first cycle is completed, thesolution of feed to be treated is injected at column 1.5.

Sub-sequence 2.1 of sequence 2 can therefore begin; note thatsub-sequence 2.1 of sequence 2 is similar to sub-sequence 1.1 ofsequence 1 except that the lines are shifted by one column.

It therefore appears that the asynchronous displacement of the linesmakes it possible to carry out, in sequence 1 on column 2, the stages ofelution, regeneration and equilibration, in fact this makes it possibleto reduce the number of columns relative to a process using adisplacement of synchronous type.

It also appears that, owing to the invention, the transition sequencecorresponding to stoppage of injection of the solution of the feed to betreated (stage (b)) makes it possible to avoid losing the product ofinterest still contained in column 4 at the end of sequence 1.4.

It is also possible to use several lines of eluents, for example fortreating solutions containing several products of interest, which aredesorbed or exchanged under different conditions. A first eluent willprovide selective recovery of the first product of interest while asecond eluent allows selective recovery of the second product ofinterest. An example of application is the recovery of milk proteins.

In the description of the present invention, the term “column” must beunderstood as meaning a physical column or any other part of a columnidentifiable as a cell, when the physical column has injection andwithdrawal points at several levels. A single physical column can thusbe divided into several sections or cells, and the invention will beapplicable to this configuration.

The invention therefore applies to all products of interest that can beseparated by chromatography. For example, the invention permits theseparation of an amino acid such as lysine, the stationary phase beingan ion exchange resin and the rinsing solutions being water. In the caseof biomolecules, for the separation of immunoglobulin (e.g. bovine,rabbit or human IG) from serum (bovine, rabbit, or human) it is possibleto use stationary phases based on the principle of affinity, for examplea Protein A grafted Sepharose gel, and eluents selected from PBSsolution (phosphate-buffered saline), glycine-HCl solution, HClsolution, etc.

EXAMPLES

The following examples illustrate the invention without limiting it.

Example 1 Amino Acid/Lysine

The process according to the invention applies in particular to an aminoacid, such as lysine; the invention therefore provides a process forseparating an amino acid from an aqueous solution containing such anamino acid and impurities, by passing this solution over a fixed bed ofion exchange resin, comprising at least four zones in series, means forflow of liquid being arranged between adjacent zones and between thelast and the first zones, said amino acid being selectively exchanged bycontact with said ion exchange resin and at least one of the impuritiesbeing exchanged relatively less on this ion exchange resin than theamino acid, the exchange capacity of said ion exchange resin beingregenerated by the action of a regenerating agent, characterized in thatit comprises several sequences, each sequence comprising the followingstages:

(a) introduction of a certain volume of water at the inlet of the firstzone and approximately simultaneous withdrawal of the same volume of aliquid diluted with said amino acid, at a point situated downstream ofthis zone;

(b) introduction of a certain volume of said aqueous solution at theinlet of the second zone and approximately simultaneous withdrawal ofthe same volume of a liquid rich in the impurity or impurities exchangedrelatively less, at a point situated downstream of this zone;

(c) introduction of a certain volume of water at the inlet of the thirdzone and approximately simultaneous withdrawal of the same volume of aliquid diluted with regenerating agent, at a point situated downstreamof this zone;

(d) introduction of a certain volume of regenerating agent at the inletof the fourth zone and approximately simultaneous withdrawal of the samevolume of a liquid rich in the amino acid, at a point situateddownstream of this zone;

it being possible for stages (a), (b), (c) and (d) to be implementedsimultaneously or not;

each subsequent sequence being carried out by the periodical downstreamdisplacement, by approximately the same volume increment, of theintroduction and withdrawal points,

and comprising moreover a stage

(e) displacement of the fronts in the zones before the periodicaldisplacement.

In the case of lysine, the fluids are culture broths, acid solutions andas regenerating agent a solution of ammonia. If the process according tothe invention is compared with a continuous process of the prior art,for example the AST process, for a given production of lysine, theprocess offers significant gains. For an identical production theconsumables are reduced by 10%, the consumption of water is slightlylower, the amount of effluent produced is approximately identical, andthe number of columns is divided by three.

Example 2 Biomolecule/IgG

For the implementation of the separation of a biomolecule, the processis implemented for example according to the procedure in FIG. 14. IgGmay be mentioned as the biomolecule. In such a case, the compositions ofthe solutions used are as follows:

Equilibration PBS (Phosphate-Buffered Saline) pH: 7.4 Renegeration HClpH: 2 Eluent Glycine HCl pH: 3 Wash PBS (Phosphate-Buffered Saline) pH:7.4 Feed Aqueous solution of immunoglobulin G IgG

The adsorption of human IgG using a ligand of the protein A type hasbeen described by Jungnauer (Journal of Chromatography B 790 (2003)35-51). The breakthrough curves of IgG on these types of stationaryphases show kinetic limitations similar to what is shown in FIG. 7. Insuch a case, the results obtained showed that the use of several columnsin the adsorption stage allows, according to the process of theinvention, better utilization of the stationary phase as shown in FIGS.7 and 8, thus obtaining a reduction in the consumption of eluent.

The process described applies quite particularly to humanimmunoglobulins G (h-IgG) or antibodies. The invention thereforeprovides a process for separating these biomolecules from a solutioncontaining said molecules as well as impurities, by passing thissolution over a bed of stationary phase specific to affinitychromatography (protein A grafted on a matrix). The affinitychromatography process developed implements the 5 chromatographic stagesdefined by the use of a specific fluid at the inlet.

The feed fluid (IgG and impurities in PBS buffer pH 7.4,Phosphate-Buffered Saline) contains the feed to be treated and itscomposition (buffer pH, salinity) makes it possible, on charging thecolumn with this fluid, for the molecule of interest to bind to thestationary phase, whereas most of the impurities pass through, withoutbeing retained. At the end of the feeding stage, the column contains astationary phase on which the component of interest is adsorbed.

The rinsing fluid (PBS buffer pH 7.4), with salinity and pH identical tothe feed fluid, does not contain feed to be treated. This stage renewsthe liquid phase of the column so that there are no longer impuritiesthat could be eluted at the same time as the IgG. This stage also avoidslosing the product of interest contained in the liquid phase of thecolumn.

The eluting fluid of the IgGs (Glycine HCl 0.1 M, pH 2.5): fluid foraltering the nature of the interactions between the IgGs and thestationary phase, it permits desorption of the target molecule from thestationary phase, the target molecule is then collected from theoutgoing liquid.

The regenerating fluid (Glycine HCl 0.1M, pH 2): after elution,impurities may remain strongly absorbed on the stationary phase, whichmay have an adverse effect on its stability or hygienic character.

The equilibration fluid (PBS buffer pH 7.4): generally identical to therinsing fluid used after feeding, this fluid makes it possible to removethe regenerating fluid from the column before carrying out the nextfeed.

The test presented according to the sequential multicolumn separationprocess uses 3 columns in this example. The 5 stages to be used in thepurification of the IgGs are distributed over the different sequencesand sub-sequences according to the diagram shown in FIG. 15.

The system made up of 3 columns implies that one cycle is divided into 3sequences. Each of the 3 sequences is divided into 4 sub-sequences:

Sequence 1:

Sub-sequence 1.1: The first two columns 1 and 2, in series, define afeed zone. Column 3 is located in the equilibration zone.

Sub-sequence 1.2: Once column 1 is almost fully charged with IgG, itpasses to the washing zone. Column 3 then enters the feed zone, the 3columns are thus put in series during this sub-sequence.

Sub-sequence 1.3: Column 1, once saturated with IgG and washed of itsimpurities, can enter the elution zone. Feed takes place on columns 2and 3 in series.

Sub-sequence 1.4: Column 1 is eluted and then regenerated. Feed stilltakes place on columns 2 and 3 in series.

For this example with 3 columns, 3 sequences are used for carrying out acomplete cycle. Sequence 2 is derived from sequence 1 by displacement ofone column of the lines of entering and leaving fluids as described inFIG. 15. Similarly, sequence 3 (not shown) is derived in an identicalmanner from sequence 2. Each of the subsequent cycles starts again fromsequence 1 and thus ensures continuity of the process.

The sequence shown in FIG. 15 comprises 5 zones, each of these zonesbeing defined by the inlets of the different fluids. For example, insub-sequence 1.1, the feed zone is located between the feed inlet oncolumn 1 and the inlet for the equilibration fluid on column 3.

In sub-sequence 1.2, where the 3 columns are in series, there isinterruption of feed and displacement of the feeding front on columns 2and 3.

In the system described previously, 2 tests were carried out at twodifferent feed rates: 450 cm/h and 620 cm/h. The feed solution containshuman polyclonal Immunoglobulins G at 1 g/l as well as impuritiescharacteristic of a feed obtained from a fermentation process. Theprocess parameters used for the purification of these IgGs are listed inTable 1.

TABLE 1 parameters used on 3 columns (1.6 cm ID × 10 cm H) containing 20ml of MabSelect Protein A phase (GE Healthcare). Sub- Sub- Sub- Sub-Sequence Cycle Rate sequence 1 sequence 2 sequence 3 sequence 4 durationduration 450 cm/h 45 min 5 min 6 min 5 min 61 min 183 min 620 cm/h 28min 5 min 7 min 5 min 45 min 135 min

The results are compared with the performances obtained on a batchcolumn (Table 2) containing the same quantity of stationary phase,namely 3×20 ml=60 ml (2.8 cm ID×10 cm H).

For identical purity and yield in batch mode and according to theprocess described, the following comparison criteria were established:

-   -   Working capacity: Quantity (in grams) of IgG fixed per litre of        stationary phase.    -   Productivity: Quantity (in grams) of IgG purified per day and        per litre of stationary phase.    -   Consumption: Total quantity of fluids used (for rinsing,        elution, regeneration, equilibration) in ml per gram of IgGs        purified.

TABLE 2 comparative results of the performances obtained in batch modeand according to the process described. Batch Mode Invention 1 column 3columns Rate Working capacity: 20 g/l 40 g/l 450 Productivity: 220 g/day· litre 310 g/day · litre cm/h consumption: 1500 ml/g 830 ml/g RateWorking capacity: 15 g/l 40 g/l 600 Productivity: 240 g/day · litre 445g/day · litre cm/h consumption: 2000 ml/g 685 ml/g

In the chromatographic processes for adsorption of antibodies carriedout in batch mode, the feed rate is relatively low in order to obtain ahigher working capacity, with the phase under consideration. Conversely,if a higher feed rate is chosen, this leads to a significant drop incapacity.

The results obtained with the process described showed that the use ofseveral columns in series in the adsorption stage provides, according tothe process described, better utilization of the stationary phase. Thus,even for high feed rates, by putting the columns in series it ispossible to operate at a capacity close to the maximum capacity of thephase.

In contrast to the batch process, this process has the advantage that itis not limited by the rate or by the working capacity. This advantagetranslates into significantly higher productivity of the processrelative to the batch process. In our example at 600 cm/h theproductivity almost doubled on passing from the batch process to theprocess described. Moreover, better utilization of the stationary phasein the process described leads to a large reduction in the consumptionof the different fluids. In our example at 600 cm/h the consumption fellalmost by a factor of 3 on passing from the batch process to the processdescribed.

Example 3 Peptide/Insulin

The process described applies quite particularly to polypeptides such ashuman insulin. The invention therefore provides a process for separatingthis polypeptide of 5.8 kDa from a solution containing said molecule aswell as impurities, by passing this solution over a bed of stationaryphase specific to ion exchange chromatography. The process of ionexchange chromatography developed on a cationic resin implements 3chromatographic stages defined by the use of a specific fluid at theinlet:

The feed fluid (Recombinant human insulin and impurities in phosphatebuffer 40 mM at pH 2.5) contains the feed to be treated. The compositionof this fluid makes it possible for the molecule of interest to bind tothe stationary phase whereas most of the impurities pass through, asthey are poorly retained. At the end of the feeding stage, the columncontains a stationary phase on which the component of interest is fixedby ionic force.

The rinsing fluid (phosphate buffer 40 mM pH 2.5) makes it possible toreplace the liquid phase of the column containing the feed with buffer.The rinsing stage thus renews the liquid phase of the column so that theimpurities contained in the liquid phase are not eluted at the same timeas the insulin during the elution stage.

The insulin eluting fluid (NaCl 1M, pH 6): This fluid will permitdesorption of the insulin from the stationary phase owing to theincrease in ionic strength and pH. The target molecule is then collectedfrom the outgoing liquid.

The test presented according to the multicolumn sequential separationprocess uses 4 columns in this example. The 3 stages (feed, rinsing,elution) to be used for the purification of insulin are distributed overthe different sequences and sub-sequences according to the diagram shownin FIG. 16.

The system made up of 4 columns means that a cycle is divided into 4sequences. Each of the 4 sequences is divided into 3 sub-sequences:

Sequence 1:

Sub-sequence 1.1: The first 3 columns, in series, define a feed zone.Column 4 is located in the elution zone.

Sub-sequence 1.2: In this sub-sequence, as column 1 is almost fullycharged with insulin, it passes to the rinsing zone. Column 4, eluted,no longer contains insulin, so it enters the feed zone, the 4 columnsare thus put in series initially. At that moment there is interruptionof feed.

Sub-sequence 1.3: The rinsing stage is carried out solely on column 1,which no longer contains insulin in the liquid phase, making it possibleto remove the poorly retained impurities. Feed is then resumed oncolumns 2, 3 and 4 that are still in series.

For this example with 4 columns, 4 sequences are to be used for carryingout a complete cycle. Sequence 2 is derived from sequence 1 by adisplacement of one column of the lines of entering and leaving fluidsas described in FIG. 16.

Thus, sequence 2 begins because column 1, saturated with insulin andrinsed of its impurities, can enter the elution zone. Feed still takesplace on columns 2, 3 and 4 in series.

Similarly, sequence 3 (not shown) is derived in an identical manner fromsequence 2. Sequence 4 (not shown) is also derived from sequence 3.

Each of the subsequent cycles starts again from sequence 1 and thusensures continuity of the process.

The sequence, shown in FIG. 16, comprises 3 zones (feed, rinsing,elution), each of these zones being defined by the inlets of thedifferent fluids. For example, in sub-sequence 1.1, the feed zone islocated between the feed inlet on column 1 and the inlet of the elutingfluid on column 4.

In sub-sequence 1.2, where the 4 columns are in series, there isinterruption of feed and displacement of the feeding front on columns 2,3 and 4.

For the system described previously, a test was simulated on the basisof experimental data at the feed rate of 600 cm/h. The feed solutioncontains human insulin at 2 g/l as well as impurities characteristic ofa feed obtained from a fermentation process. The process parameters usedfor the purification of this insulin solution are listed in Table 1.

TABLE 1 Parameters used on 4 columns (ID: 0.5 cm × H: 10 cm) eachcontaining 2 ml of cationic resin Toyopearl SP 550 C (Tosoh) LinearDuration of Duration of Duration of Se- flow Sub- Sub- Sub- quence Cyclerate sequence 1 sequence 2 sequence 3 duration duration 600 cm/h 45 min5 min 10 min 60 min 240 min

The results are compared with the performances that would be obtained ona batch column (Table 2) containing the same amount of stationary phase,namely 4×2 ml=8 ml (1 cm ID×10 cm H).

For identical purity and yield in batch mode and according to theprocess described, the following comparison criteria were established:

Working capacity: Quantity (in grams) of insulin fixed per litre ofstationary phase.

Productivity: Quantity (in grams) of Insulin purified per day and perlitre of stationary phase.

Consumption: Total quantity of fluids used (rinsing, elution) in ml pergram of insulin purified.

TABLE 2 Comparative results of the performances obtained in batch modeand according to the process described. Batch Invention 1 column 4columns Rate Working capacity: 40 g/l 110 g/l 600 Productivity: 640g/day · litre 660 g/day · litre cm/h Consumption of 1750 ml/g 500 ml/gbuffer:

In the chromatographic ion exchange adsorption processes carried out inbatch mode, the feed rate is relatively low in order to obtain, on thephase under consideration, a higher working capacity, which is almosttripled. Conversely, if a higher feed rate is selected, this leads to asignificant drop in capacity.

The results obtained according to the process described showed that theuse of several columns in series in the adsorption stage provides,according to the process described, better utilization of the stationaryphase. Thus, even for high feed rates, it is possible, by connecting thecolumns in series, to operate at a capacity close to the maximumcapacity of the phase.

In contrast to the batch process, this process has the advantage of notbeing limited by the rate nor by the working capacity. This advantagetranslates into higher productivity of the process relative to that ofthe batch process as well as a large reduction in consumption of thedifferent fluids. In our example at 600 cm/h, for equivalentproductivity in batch mode and according to the process described, theconsumption of the expensive buffers was divided almost by a factor of 3on changing from the batch process to the process described. Tests athigher rates would show even more clearly the differences inproductivity and consumption between the process described and the batchprocess.

In addition, we compared our process with other processes havingcontinuous injection of feed, such as SMB for example, which does notallow operating with a reduced number of columns with several eluentsand without loss of yield. In our example, the presence of sub-sequence2 without feed injection makes it possible to gain 2 to 10% of yielddepending on the parameters of feed composition.

1. Process of separation on a solid support by selective sequentialmulticolumn retention for separating a product of interest from asolution containing said product of interest, by passing this solutionover a fixed bed of chromatographic resin comprising at least threezones, means for flow of liquid being arranged between adjacent zonesand between the last and the first zone, said process comprising severalsequences, each sequence comprising at least one stage selected from anadsorption stage, a rinsing stage, a desorption stage, implementedsimultaneously or not simultaneously, each subsequent sequence beingcarried out by the downstream displacement of the fronts in the zones byapproximately the same increment before the periodical displacement ofthe introduction and withdrawal points, wherein the process comprises asub-sequence without feed injection wherein the process comprisesseveral sequences, each sequence comprising at least one of thefollowing stages: (a) introduction of a certain volume of a rinsingsolution at the inlet of the first zone and approximately simultaneouswithdrawal of the same volume of a liquid diluted with said product ofinterest, at a point situated downstream of this zone; (b) introductionof a certain volume of said feed solution at the inlet of the secondzone and approximately simultaneous withdrawal of the same volume of aliquid rich in the impurity or impurities that are retained relativelyless, at a point situated downstream of this zone; (c) introduction of acertain volume of a rinsing solution at the inlet of the third zone andapproximately simultaneous withdrawal of the same volume of a liquiddiluted with regenerating agent, at a point situated downstream of thiszone; (d) introduction of a certain volume of regenerating agent at theinlet of the fourth zone and approximately simultaneous withdrawal ofthe same volume of a diluted liquid, at a point situated downstream ofthis zone; (e) introduction of a certain volume of an eluent at theinlet of the fifth zone and approximately simultaneous withdrawal of thesame volume of a liquid rich in said product of interest, at a pointsituated downstream of this zone; it being possible for stages (a), (b),(c), (d) and (e) to be implemented simultaneously or not simultaneously;each subsequent sequence being carried out by the periodical downstreamdisplacement, by approximately the same volume increment, of theintroduction and withdrawal points, and comprising moreover a stage (f)displacement of the fronts in at least zone (b) before the periodicaldisplacement.
 2. Process according to claim 1, wherein stages (d), and(e) are implemented with the same fluid, said stages then correspondingto a stage consisting of: (d) introduction of a certain volume ofregenerating agent at the inlet of the fourth zone and approximatelysimultaneous withdrawal of the same volume of a liquid rich in saidproduct of interest, at a point situated downstream of this zone; thefourth and fifth zones then being merged in a single fourth zone. 3.Process according to claim 1, wherein stages (a), (b), (c) and (d) areimplemented at least partly simultaneously.
 4. Process according toclaim 1, wherein said displacement of the fronts displaces the frontssynchronously in the different zones.
 5. Process according to claim 4,wherein the displacement of the fronts comprises the following stages:(i) creation of a circulation loop zone between the different zones; and(ii) applying circulation in said loop for displacing the fronts. 6.Process according to claim 4, wherein the displacement of the frontscomprises the following stages: (i) creation of a first displacementzone by fluid connection from the outlet of the first zone to the inletof the second zone and by fluid connection from the outlet of the secondzone to the inlet of the third zone, and downstream displacement of theinlet of the first zone to provide the inlet of the first displacementzone and upstream displacement of the outlet of the third zone toprovide the outlet of the first displacement zone; and creation of asecond displacement zone by fluid connection from the outlet of thethird zone to the inlet of the fourth zone and by fluid connection fromthe outlet of the fourth zone to the inlet of the fifth zone and fluidconnection from the outlet of the fifth zone to the inlet of the firstzone, and downstream displacement of the inlet of the third zone toprovide the inlet of the second displacement zone and upstreamdisplacement of the outlet of the first zone to provide the outlet ofthe second displacement zone; and (ii) introduction of a certain volumeof rinsing solution at the inlet of the first displacement zone andapproximately simultaneous withdrawal of the same volume of rinsingsolution recovered at the outlet of the first displacement zone; (iii)introduction of a certain volume of rinsing solution at the inlet of thesecond displacement zone and approximately simultaneous withdrawal ofthe same volume of rinsing solution recovered at the outlet of thesecond displacement zone.
 7. Process according to claim 1, wherein saiddisplacement of the fronts displaces the fronts asynchronously in thedifferent zones.
 8. Process according to claim 7, wherein thedisplacement of the fronts comprises the following stages: (i) creationof a first zone of a first displacement by fluid connection from theoutlet of the first zone to the inlet of the second zone and by fluidconnection from the outlet of the second zone to the inlet of the thirdzone; and creation of a second zone of a first displacement by fluidconnection from the outlet of the third zone to the inlet of the fourthzone and by fluid connection from the outlet of the fourth zone to theinlet of the fifth zone and by fluid connection from the outlet of thefifth zone to the inlet of the first zone; and (ii) introduction of acertain volume of said solution at the inlet of the first displacementzone and approximately simultaneous withdrawal of the same volume of aliquid diluted with regenerating agent at the outlet from the first zoneof a first displacement; (iii) introduction of a certain volume ofregenerating agent at the inlet of the second displacement zone andapproximately simultaneous withdrawal of the same volume of a liquiddiluted with said product of interest at the outlet from the second zoneof a first displacement; (iv) creation of a first zone of a seconddisplacement by fluid connection from the outlet of the first zone tothe inlet of the second zone and by fluid connection from the outlet ofthe second zone to the inlet of the third zone, and downstreamdisplacement of the inlet of the first zone to provide the inlet of thefirst zone of a second displacement and upstream displacement of theoutlet of the third zone to provide the outlet of the first zone of asecond displacement; and creation of a second zone of a seconddisplacement by fluid connection from the outlet of the third zone tothe inlet of the fourth zone and by fluid connection from the outlet ofthe fourth zone to the inlet of the fifth zone and fluid connection fromthe outlet of the fifth zone to the inlet of the first zone, anddownstream displacement of the inlet of the third zone to provide theinlet of the second displacement zone and upstream displacement of theoutlet of the first zone to provide the outlet of the seconddisplacement zone; and (vi) introduction of a certain volume of rinsingsolution at the inlet of the first zone of a second displacement andapproximately simultaneous withdrawal of the same volume of a liquidrich in the impurity or impurities that are retained relatively less atthe outlet from the first zone of a second displacement. (vii)introduction of a certain volume of rinsing solution at the inlet of thesecond zone of a second displacement and approximately simultaneouswithdrawal of the same volume of a liquid rich in said product ofinterest at the outlet from the second zone of a second displacement. 9.Process according to claim 1, wherein stage (f) comprises a displacementof the fronts in all the zones before the periodical displacement. 10.Process according to claim 1, wherein the volume increment according towhich said introduction points and said withdrawal points are displacedcorresponds approximately to the volume of an entire fraction of a zoneof absorbent material.
 11. Process according to claim 1, wherein theperiodical displacement of the stages is synchronous.
 12. Processaccording to claim 1, wherein the periodical displacement of the stagesis asynchronous.
 13. Process according to claim 1, wherein said liquiddiluted with said product of interest is sent at least partly to stage(b).
 14. Process according to claim 1, wherein there is a supplementaryzone, and in that it additionally comprises a stage (g) of introductionof all or part of the liquid diluted with said product of interestobtained in stage (a) in said supplementary zone, and recovery ofapproximately the same volume of rinsing solution at a point situateddownstream of this zone.
 15. Process according to claim 1, wherein stage(b) comprises two substages (b1) and (b2), as well as an intermediatestage of adjustment of a parameter of the solution, in particular bymodification of the pH.
 16. Process according to claim 1, wherein saidliquid diluted with regenerating agent obtained in stage (c) is sent atleast partly to stage (d), optionally after being topped up.
 17. Processaccording to claim 1, wherein the recovered rinsing solutions are sentat least partly to stages (a) and/or (c).
 18. Process according to claim1, wherein the chromatography is an ion exchange chromatography and theproduct of interest is an amino acid, preferably lysine.
 19. Processaccording to claim 1, wherein the chromatography is an ion exchangechromatography and the product of interest is insulin.
 20. Processaccording to claim 1, wherein the chromatography is an affinitychromatography and the product of interest is IgG.
 21. Process accordingto claim 1, wherein the chromatography is an affinity chromatography andthe product of interest is a protein or a biomolecule.